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Abstract

Introduction

The present study aimed to explore a possible role for IL-21 producing Th-cells in
the immunopathogenesis of granulomatosis with polyangiitis (GPA).

Methods

Peripheral blood from 42 GPA patients in remission and 29 age-matched healthy controls
(HCs) were stimulated in vitro, and the frequencies of IL-21 producing Th-cells were determined by flow cytometry.
Since Th17-cells produce a low level of IL-21, IL-17 was also included in the analysis.
Given that IL-21 is a hallmark cytokine for T follicular helper cells (TFH), we next evaluated the expression of their key transcription factor BCL-6 by RT-PCR
and flow cytometry. To investigate the effect of IL-21 on autoantibody-production,
PBMCs from GPA patients were stimulated in vitro with BAFF/IL-21 and total IgG and ANCA levels were measured in supernatants. In addition,
the expression of IL-21-receptor on B-cells was analyzed.

Results

Percentages of IL-21 producing Th-cells were significantly elevated in GPA-patients
compared to HCs, and were restricted to ANCA-positive patients. The expression of
BCL-6 was significantly higher in ANCA-positive GPA-patients, as compared with ANCA-negative
patients and HCs. IL-21 enhanced the production of IgG and ANCA in vitro in stimulated PBMCs from GPA patients. No difference was found in the expression of
the IL-21-receptor on B-cells between ANCA-negative patients, ANCA-positive patients,
and HCs.

Conclusion

The increased frequency of circulating IL-21 producing Th-cells in ANCA-positive GPA
patients and the stimulating capacity of IL-21 on ANCA-production suggest a role for
these cells in the immunopathogenesis of GPA. Blockade of IL-21 could constitute a
new therapeutic strategy for GPA.

Introduction

Granulomatosis with polyangiitis (GPA) is an autoimmune vasculitis of small- to medium-sized
blood vessels, associated with the presence of circulating anti-neutrophil cytoplasmic
autoantibodies (ANCA) that are mainly directed against proteinase 3 [1-3]. Histopathologically, GPA is characterized by granulomatous inflammation and pauci-immune
vasculitis, including necrotizing crescentic glomerulonephritis.

Although the production of ANCA is directly attributable to autoreactive B-cells,
there is extensive evidence that T-cells play a critical role in GPA as well. The
immunoglobulin (Ig)G subclass distribution of ANCA, with a preponderance of the IgG1
and IgG4 subclasses, suggests a T-cell-dependent immune response [4]. Infiltrating T-cells in granulomatous lesions and persistent T-cell activation have
been observed in GPA patients [5,6]. In addition, an aberrant T-cell phenotype and impaired regulatory T-cell function
are also reported in GPA patients in remission [7-9], suggesting that even during remission, the immune system is dysregulated. Moreover,
T-helper (Th) cell polarization with an increase in Th17 cells has been demonstrated
[10,11]. Th17 cells and their cytokine IL-17 have been shown to play a critical role in many
inflammatory diseases. In addition to IL-17, Th17 cells can produce IL-21, a cytokine
that is largely responsible for B-cell class switching and antibody production, and
which induces differentiation of B-cells towards plasma cells by synergizing with
B-cell activating factor (BAFF)[12,13]. Therefore, it is conceivable that IL-21 may contribute to the production of pathogenic
autoantibodies in GPA.

Multiple studies in animal models indicate a pivotal role of IL-21 in the pathogenesis
of autoimmune diseases. Studies in arthritis models have shown that blockade of IL-21
activity reduces joint inflammation and destruction [14]. Subsequent investigations demonstrated that blocking of the IL-21 pathway reduces
levels of anti-dsDNA autoantibodies and prevents renal disease in mouse models of
systemic lupus erythematosus (SLE) [15]. In addition, mice deficient in IL-21-receptor expression were found to be protected
to a large extent against the development of inflammatory bowel disease (IBD) and
type-I diabetes [16,17]. Interestingly, recent genome-wide association studies have provided convincing evidence
that genetic variants in the region on chromosome 4q27 that harbor the IL-21 and IL-2
genes are associated with chronic inflammatory disorders, including SLE, IBD and psoriasis
[18-20]. Thus, IL-21 seems to play an important role in autoimmune diseases in general and
could constitute a novel target for therapy.

IL-21 is produced mainly by activated CD4+ Th-cells. Recent studies have demonstrated that IL-21, besides its production by Th17
cells, is predominantly secreted by a distinct Th-cell lineage, termed follicular
helper T-cells (TFH) that express the transcription factor BCL-6 and are considered to be specialized
providers of B-cell help [21]. Expansion of circulating T-cells resembling TFH cells has been reported in patients with SLE and in patients with rheumatoid arthritis
[22-24]. To date, no study has investigated the role of IL-21-producing Th-cells in GPA.
Therefore, this study aimed to assess the frequency of IL-21-producing Th-cells, and
to evaluate whether TFH cells or Th17 cells are the major source of IL-21 in GPA patients. For this purpose,
we examined the expression of both IL-21 and IL-17 in circulating CD4+ T-cells of patients with GPA. To improve our understanding of the role of IL-21-producing
Th cells in autoantibody production we assessed their frequencies in ANCA-positive
and ANCA-negative patients, and studied effects of IL-21 on Ig and ANCA production
in vitro.

Methods

Study population

Forty-two patients with GPA and 29 age- and sex-matched healthy controls (HC) (18
male, 11 female, mean age 56 (SD ± 13) years, range 26 to 72 years, P = 0.16) were included in this study. The diagnosis of GPA was established according
to the definitions of the Chapel Hill Consensus Conference and patients fulfilled
the classification criteria of the American College of Rheumatology (ACR)[25,26]. Only patients without clinical signs and symptoms of active vasculitis and considered
to be in complete remission, as indicated by a score of zero on the Birmingham Vasculitis
Activity Score (BVAS), were included in the study [27]. Serostatus for ANCA was followed for several months in all patients, and patients
with a stable serostatus for ANCA (positive or negative) for at least 3 months were
included in this study. Based on these criteria, 23 patients were positive for PR3-ANCA,
whereas 19 were ANCA-negative. There was history of generalized disease including
renal involvement in 27 patients, and 15 patients had localized disease, which had
been confined to the upper and/or lower respiratory tract. None of the patients and
controls had infection at the time of sampling. Eleven GPA-patients (eight ANCA-positive,
and three ANCA-negative) were treated with maintenance immunosuppressive therapy at
the time of blood sampling. Four of them were treated with only azathioprine (25 to
100 mg/day), one patient with mycophenolate mofetil (1500 mg/day), and six patients
received prednisolone (5 to 10 mg/day) in combination with azathioprine (125 mg/day).
Participants in rituximab trials were excluded from the present study. The main clinical
and laboratory data from the patients are summarized in Table 1. All patients and healthy individuals provided informed consent and the study was
approved by the local Medical Ethics Committee of the University Medical Centre Groningen,
University of Groningen (NL).

Table 1. Clinical and laboratory characteristics of patients with granulomatosis with polyangiitis
(GPA) at the time of blood sampling

Measurement of ANCA titres and specificity

ANCA titers were measured by indirect immunofluorescence (IIF) on ethanol-fixed human
granulocytes according to standard procedures as previously described [28]. ANCA titers higher than 1:40 were considered positive. ANCA antigenic specificity
was determined using an in-house capture ELISA as described before [29,30]. Briefly, a 96-well plate was coated with goat-anti-mouse Ig for 48 hours. After
washing, plates were incubated with mouse monoclonal antibody against human PR3 for
2 hours. After washing, the plate was incubated overnight at 4°C with an extract of
human azurophilic granules, which were isolated from neutrophils of healthy donors.
Further, serial dilutions of serum (with a starting dilution of 1:100) were incubated
for 1 hour. The plate was washed, and the captured antibodies were detected with purified
F(ab)2 goat-anti-human IgG conjugated to alkaline phosphatase. P-nitrophenyl-phosphate disodium
was used as a substrate, and the optical density was measured at 405 nm.

After stimulation cells were washed in wash buffer (PBS, 5% fetal bovine serum (FBS),
0.1% sodium azide (Merk, Germany)] and stained with PerCP-conjugated anti-CD8 and
APC-conjugated anti-CD3 for 15 minutes at room temperature. Cells were fixed with
100 μL Reagent A (Caltag/Invitrogen., Breda, The Netherlands) for 10 minutes. After
washing, the pellet was resuspended in 100 μL permeabilization Reagent B (Caltag/Invitrogen)
and labeled with A488-conjugated anti-IL-17 and PE-conjugated anti-IL-21 for 20 minutes
in the dark. After staining, the cells were washed and immediately analyzed on the
FACS-Calibur flow cytometer (Becton & Dickinson). Data were collected for 2 × 105 cells, and plotted using the Win-List software package (Verity Software House Inc,
ME, USA) ME, USA). Because stimulation reduces surface expression of CD4 on T-cells,
CD4+T-cells were identified indirectly by gating on CD3-positive and CD8-negative lymphocytes.
Gated CD4+ T-cells were further displayed as a dot plot for evaluation of intracellular cytokine
production. The unstimulated control sample was used as a guide for setting the linear
gates to discriminate positive and negative populations.

Intracellular staining for transcription factors

Peripheral blood mononuclear cells (PBMCs) from GPA patients and HCs were prepared
from heparinized venous blood by density-gradient centrifugation on Lymphoprep (Axis-Shield
PoC AS, Oslo, Norway). Cells recovered from the gradient interface were washed twice
in PBS and stained for BCL-6 and FoxP3 according to the manufacturer's instructions
(eBioscience staining set for transcription factors). Briefly, PBMCs were adjusted
to 1 × 106 cells in 100 μL and incubated with appropriate concentration of APC-conjugated anti-CD3
and PerCP-conjugated anti-CD8 for 30 minutes at 4°C in the dark, followed by fixation
and permeabilizaion in Fix/Perm buffer (eBioscience) for 45 minutes. Cells were then
washed twice with 1 × permeabilization buffer (eBioscience), and stained with PE-conjugated
anti-BCL-6 and A488-conjugated anti-FoxP3. After incubation for 30 minutes in the
dark, the cell suspension was washed and immediately analyzed on the FACS-Calibur
flow cytometer (Becton & Dickinson). Lymphocytes were gated by forward and side scatter
patterns, and plotted using the Win-List software package (Verity Software House Inc).
Isotype matched control antibodies of irrelevant specificity were obtained from eBioscience
and R&D systems.

Immunofluorescent surface staining for IL-21R on B-cells

Fresh blood samples from GPA patients and HCs were labeled with PE-conjugated anti-IL21R,
and PerCP-conjugated anti-CD19 for 15 minutes in the dark. Cells were successively
treated with 2 mL diluted FACS lysing solution (BD, Amsterdam, The Netherlands) for
10 minutes and then washed twice in wash buffer and immediately analyzed by flow cytometry.

RNA isolation and real-time reverse transcription (RT)-PCR

Erythrocytes were lysed and leukocytes were fixed and washed twice in 1% BSA. RNA
was isolated from total leukocytes with TRIzol reagent (Invitrogen) according to the
manufacturer's instructions. DNAse treatment (Ambion, Huntingdon, Cambridgeshire,
UK) was performed and subsequently cDNA was synthesized using M-MLV reverse transcriptase
and oligo (dT) 14 to 18 as primer. For measurement of mRNA for BCL-6 and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), 1 μL of cDNA in triplicate was used for amplification by the
Taqman RT-PCR system (ABI Prism 7900HT Sequence Detection System, Applied Biosystems,
Foster City, CA, USA) with specific Taqman primers/probes (BCL-6 (Hs 00153368_m1)
and GAPDH (Hs 99999905_m1), Applied Biosystems). Amplification was performed using
standard conditions and calculations of fold induction were performed. We normalized
gene expression to GAPDH and expressed values relative to control using the ΔΔCT (cycle threshold) method.

Measurement of in vitro production of PR3-ANCA

In vitro PR3-ANCA IgG production in PBMC culture supernatants was measured by Phadia ImmunoCAP® 250 analyzer (Thermo Fisher Scientific, MA, USA) using ELiA™ PR3, and the levels of
PR3-ANCA IgG production were expressed in response units (RU).

Statistical analysis

Data are presented as median values unless stated otherwise. The nonparametric Mann-Whitney
U-test was used to compare data from patients with those of HCs. The Wilcoxon matched
pairs test was used for intra-individual comparison. Correlations were assessed using
Spearman's rank correlation coefficient. Two-tailed P-values lower than 0.05 were considered statistically significant.

Results

We initially determined the frequency of IL-21-producing CD4 T-cells in the peripheral
blood of GPA patients (n = 42) and HCs (n = 29) after in vitro stimulation. The percentage of circulating IL-21+ Th-cells was significantly higher in GPA patients compared with the control group
(Figure 1B). Of note, Th17 cells may produce IL-21 in addition to their signature cytokine IL-17.
Since Th17 cells are increased in GPA patients [10,11], we next extended our analysis to investigate whether increased IL-21+ Th-cells in GPA patients resulted from an increase in Th17 cells. To this end, IL-17
staining was included in the analysis to determine what percentage of the total IL-21+ Th-cells are Th17 cells. Using this approach, we assessed the frequency of IL-21+IL-17-, IL-21+IL-17+, and IL-21-IL-17+ cells within the CD4 T-cells in GPA patients and HCs. As shown in Figure (1C, D and 1E), GPA patients in remission had a significantly higher percentages of circulating
IL-21+IL-17-, IL-21+IL-17+, and IL-21-IL-17+ cells compared with the control group. However, the majority of circulating CD4+ T-cells that produced only IL-21 were distinct from Th17 cells, that is, negative
for IL-17. To assess the possible role of IL-21+IL-17- Th-cells in ANCA production, we compared their percentage between patients who were
ANCA-positive (n = 23; IIF titer >1:40) or ANCA-negative (n = 19) at the time of inclusion. Significant increases in the frequencies of IL-21+IL-17- Th-cells were observed in ANCA-positive patients in comparison with HCs and ANCA-negative
patients, whereas no significant difference was found between ANCA-negative patients
and HCs (Figure 1F). In contrast, the percentages of IL-21+IL-17+ and IL-21-IL-17+ Th-cells in ANCA-positive GPA patients did not differ from those in ANCA-negative
GPA-patients (Figure 1G and 1H). These results suggest that persistence of IL-21+IL-17- Th-cells during remission plays a role in the ongoing humoral autoimmune response
in ANCA-positive GPA patients.

To rule out the possibility that the increased proportion of IL-21+IL-17- Th-cells in GPA patients was the result of current treatment, the ANCA-positive patient
group was divided into treated and untreated patients, and the percentages of IL-21+IL-17- Th-cells were compared. No significant differences were observed between treated and
untreated patients (data not shown). We also compared the percentage of IL-21+IL-17- Th-cells between currently untreated ANCA-positive patients with a history of generalized
disease and those with localized disease. No difference was found between these patient
groups (data not shown).

It has been reported that IL-21 is a key factor regulating the differentiation of
naïve CD4+ T-cells into Th17 cells [32,33]. In order to analyze this relationship, we tested correlation between percentages
of IL-21+IL-17- Th-cells and percentages of terminally differentiated Th17 cells (IL-21-IL-17+) in GPA patients (n = 42) and HCs (n = 29). Interestingly, a significant positive correlation was observed between IL-21+IL-17- Th-cells and IL-21-IL-17+ Th-cells in both GPA patients and HCs (r = 0.58, P < 0.0001 and r = 0.37, P = 0.04, respectively) (Figure 2A and 2B).

Figure 2.Correlation between the percentages of IL-21+IL-17- cells and IL-21-IL-17+ cells within the CD4+ T-cells in peripheral blood of healthy controls (HCs) (A), and patients with granulomatosis
with polyangiitis (GPA) (B). Spearman rank correlation coefficients (r) and P-values are given.

Since IL-21 is not the only marker for TFH cells, we further characterized the identity of circulating IL-21-producing cells
by analyzing BCL-6 expression, which is considered a master regulator and specific
transcription factor for TFH cells [34,35]. To this end, the expression of mRNA BCL-6 was assessed in circulating leukocytes
from GPA patients and HCs by real-time RT-PCR. Restricted numbers of patients and
controls were included in this analysis due to insufficient cell numbers. Patients
with ANCA-positive GPA (n = 10) had a significantly higher expression of mRNA BCL-6 than ANCA-negative patients
(n = 6) and HCs (n = 11) (Figure 3B). In addition, intracellular FACS-staining for BCL-6 within circulating CD4+ T cells
confirmed the increased BCL-6 expression in ANCA-positive GPA patients (Figure 3A and 3C). We have also analyzed the MFI (mean fluorescence intensity) of BCL-6 expression
in CD4+ T-cells from patients and HCs and found that the expression level of BCL-6 per Th-cell
in GPA patients was similar to that in HCs (data not shown). Thus, BCL-6 expression
is increased in GPA patients due to increased frequencies of circulating BCL-6+ CD4+ T-cells.

Because in recent studies a new population of FoxP3+ regulatory T-cells has been described that shares features with TFH cells by expressing the transcription factor BCL-6, we also evaluated whether the
increase in BCL-6+ T-cells in GPA patients was a result of an increase in FoxP3+BCL-6+ T-cells [36,37]. This analysis showed that the increase in BCL-6 expression in GPA patients was restricted
to TFH cells and although a low percentage of FoxP3+BCL-6+ T-cells was found (< 0.3%), no differences in these cell frequencies were observed
between GPA patients and HCs (data not shown).

Proportions of IL-21-receptor expressing B-cells do not differ between GPA patients
and healthy controls

Since it is well known that IL-21 acts on B-cells to support their expansion and antibody
production [38-40], we conducted further analysis to compare the expression of IL-21R on B cells from
GPA patients and HCs. No differences were seen in the percentages of IL-21R+ B-cells either between ANCA-positive (n = 13) and ANCA-negative patients (n = 14) or between patients and HCs (n = 19) (Figure 4).

IL-21 induces IgG and ANCA production by B-cells from GPA patients

To explore the interplay between IL-21-producing Th-cells and B-cells in GPA patients,
we investigated the effect of IL-21 on IgG antibody-production by B-cells from GPA
patients. Restricted numbers of patients and controls were enrolled in this analysis
due to insufficient cell numbers. PBMCs from GPA patients were cultured in vitro in the presence or absence of exogenous IL-21 for 12 days and total IgG was measured
in supernatants by ELISA. Because IL-21 promotes B-cell differentiation by synergizing
with BAFF [12,13], we questioned whether the effect of IL-21 on IgG production could be augmented by
adding BAFF to the culture. Of note, autologous T-cells in our culture system act
as a natural provider of CD40 ligation for B-cells, as this ligation is required for
B-cell activation, isotype switching and memory development. As shown in Figure 5, IL-21 significantly enhanced the production of IgG in vitro in stimulated PBMCs from both ANCA-positive (n = 7) and ANCA-negative (n = 6) GPA-patients, whereas stimulation with BAFF alone did not result in increased
IgG production. The combination of BAFF and IL-21 tended to increase IgG production
more than IL-21 alone. Next, we assessed the effect of IL-21 plus BAFF on in vitro production of PR3-ANCA. As shown in Figure 5B, spontaneous PR3-ANCA production was observed in cultured PBMCs from ANCA-positive
patients (n = 16) in comparison with cells from ANCA-negative patients (n = 12). Importantly, IL-21 induces a significant enhancement in PR3-ANCA production
in PBMCs isolated from ANCA-positive patients in comparison with ANCA-negative patients.
So it is conceivable that autoreactive B-cells were enriched in the peripheral blood
of ANCA-positive patients.

Discussion

In the present study, we demonstrate an increase in the percentage of circulating
IL-21-producing Th-cells in GPA patients. We found that elevated frequencies of IL-21-producing
Th-cells were restricted to ANCA-positive GPA patients and that these cells were distinct
from Th17-cells. We also confirmed that IL-21 can enhance the production of IgG and
ANCA in vitro.

Over the past few years, Th17-cells have challenged the classical Th1/Th2 paradigm,
and have been implicated in a growing number of autoimmune and inflammatory diseases
[41]. Recently, a distinct Th-cell subset termed TFH and characterized by elevated expression levels of multiple surface proteins and BCL-6
as well as enhanced IL-21 secretion, have been identified as true helper cells for
antibody responses. We and others have previously demonstrated that circulating Th17-cells
are significantly increased in GPA patients even during quiescent disease [10,11]. However, data are lacking to support a role of IL-21-producing Th-cells in GPA.
Since Th17 cells also produce IL-21, we investigated whether Th17 cells in GPA are
a source of IL-21. Strikingly, the majority of circulating CD4 T-cells that produced
IL-21 were distinct from Th17 cells, indicating that other Th-cell subsets such as
TFH cells are the source of this cytokine. Importantly, the expansion of TFH cells in GPA patients was confirmed by increased BCL-6 expression. To the best of
our knowledge, this is the first report demonstrating an increase in the frequency
of circulating IL-21-producing Th-cells in GPA, suggesting that TFH cell-derived IL-21 may contribute to disease pathogenesis via stimulation of (auto)antibody
production.

TFH cells are considered to be the major source of IL-21 and seem to be an important subset
for adaptive immune responses, although there are conflicting reports on their mode
of action in vivo. It has been demonstrated that IL-21-producing Th-cells induce Th17 development and
proliferation [32,33], which has been shown to promote germinal center (GC) formation in a BXD2 mouse model
of autoimmunity [42]. In agreement with these findings, we demonstrate a significant positive relationship
between IL-17+IL-21- Th-cells and IL-17-IL-21+ Th-cells in peripheral blood of GPA patients. It seems likely that increased Th17
cells in GPA-patients are the result of an enhanced TFH response, which in turn may participate in granuloma formation and vascular damage.
The role of IL-21 in vasculitis was previously suggested by Chen and coworkers [43]. In their study, mice deficient in interferon regulatory factor-4, a protein that
inhibits IL-17A production, rapidly developed large-vessel vasculitis and showed increased
IL-21 synthesis in addition to increased IL-17A production [43]. Moreover, a role of IL-21 in recruitment of Th17-cells to inflamed tissues has been
reported by Caruso and coworkers [44] by showing that IL-21 induces gut epithelial cells to secrete macrophage inflammatory
protein-3α (MIP-3α), a chemokine that mediates Th17-cell homing to the skin, joints,
and mucosal tissues. Given that endothelial cells are known to produce MIP-3α, it
is possible that IL-21 in GPA patients enhances the migration and accumulation of
Th17-cells into the vascular wall resulting in inflammation. Besides, IL-21 was shown
to enhance granzyme B expression [45] and increase perforin-mediated cytotoxicity by human CD8 T-cells [46] and natural killer cells [47]. It is therefore conceivable that IL-21 can contribute to vessel injury and disease
progression in GPA patients. This is an area worth of further investigation.

In contrast to the pro-inflammatory role of TFH cells, recent studies have identified a distinctive population of TFH cells that displays a regulatory function and suppresses the differentiation of GC
B-cells in follicles in vivo. This subset was termed follicular regulatory T-cells (TFR), which express the regulatory transcription factor FoxP3 in addition to their specific lineage transcription factor BCL-6 [36,37]. As circulating FoxP3+ T-cells are increased in GPA patients [7], it is conceivable that the observed increase in TFH cells in patients is due to an increase in TFR cells that co-express FoxP3 and BCL-6. We have investigated this possibility but found that the increase in circulating
TFH cells in GPA patients cannot be explained by increase in TFR cells (data not shown).

In our study, increased frequencies of TFH cells were observed in patients who were ANCA-positive at the time of inclusion. This
suggests the involvement of IL-21 in the process of autoantibody production in GPA.
These data are in line with previous reports showing that TFH cells act directly on B-cells through the IL-21/IL-21R pathway, and that IL-21 is
a potent inducer of class-switch recombination and plasma cell differentiation [39,48,49]. The expression of IL-21R on B-cells from ANCA-positive and ANCA-negative GPA patients
was comparable, which suggests that both patient populations have the same ability
to respond to IL-21. However, in vitro stimulation with IL-21 enhanced the production of ANCA in cell cultures from ANCA-positive
patients only, although enhanced total IgG-production was observed in both patient
groups. So it is conceivable that autoreactive B-cells were enriched in the peripheral
blood of ANCA-positive patients. This might be clinically relevant as well, because
ANCA-positive patients are at increased risk for disease relapse [50,51].

In this study, patients were evaluated for the distribution of TFH cells during remission. We have previously shown that activated T-cells are present
at the time of clinically quiescent disease [9,10]. Furthermore, during active disease effector T-cells appear to migrate towards inflamed
tissue [52]. Therefore, in order to study dysbalance of T-cells in GPA patients using peripheral
blood samples, we choose to- select patients without or with low dosages of immunosuppressive
medication and at the time of clinically quiescent disease.

Conclusions

In conclusion, the data presented here demonstrate a prominent increase of circulating
TFH cells in ANCA-positive GPA patients. The key cytokine of these TFH cells, that is IL-21, contributes to the production of ANCA autoantibodies in vitro. These data support the notion that TFH cells are associated with the pathogenic process in GPA patients and may constitute
a novel target for therapeutic intervention.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

All authors contributed to the design, acquisition of data, analysis and interpretation
of data. WHA contributed to concept and design, performed the statistical analysis,
and had full access to all of the data in the study and takes responsibility for the
integrity of the data and the accuracy of the data analysis. WHA, NL, MGH, BDvdM,
and HT performed the flow cytometry, in vitro experiments, RT-PCR experiments, interpretation of data, and drafting of the manuscript.
CAS and AR contributed to concept and design, inclusion of GPA patients, analyses
and interpretation of clinical data, and drafting of the manuscript. PCL, PH, and
CGMK contributed to concept and design, interpretation of data and revising the manuscript
for important intellectual content. All authors read and approved the final manuscript.

Acknowledgements

We are grateful to the patients and healthy donors for their co-operation and participation
in this study. The research leading to these results has received funding from the
European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n°
261382, and from the Groningen University Institute for Drug Exploration (GUIDE).